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Fab Library Construction Protocol

The antigen-binding (Fab) fragment is a region on an antibody that binds to antigens. The “fab fragment” (“fragment, antigen binding”) was originally defined as one of the cleavage products after treatment of rabbit IgG with papain, which cleaves the core hinge, resulting in two identical fab fragments and the intact Fc as products. It contains four domains: the heavy chain variable domain (VH) linked to constant domain 1 (CH1), and the light chain variable domain (VL) linked to a constant domain (CL). Fab libraries, in which light-chain (LC, contains κ chain or λ chain) genes and heavy-chain (HC, here we only illustrate γ chain) variable region genes as well as CH1 genes are cloned into a phagemid vector and subsequently displayed on the surface of the filamentous phage particle, have been widely used for the isolation of antibodies with specificity for haptens, foreign antigens, and self antigens. The isolation of Fabs from combinatorial libraries is thus valuable in contributing to the understanding of Antibody–Antigen interactions, as well as the nature of the in vivo immune response.

Compared with scFv libraries, the construction of Fab libraries has the advantage of simplicity in technically, because scfv fragments have more tendencies to form dimers and higher order multimers in a clone-dependent and relatively unpredictable way. Multimeric antibody molecules bind more strongly to immobilized antigen than monomeric fragments because of their greater avidity, and therefore have higher “apparent” affinities. This explains why an accurate determination of the affinity is not easily possible with mixtures of mono- and multimeric scFv fragments.

pComb3 Vector Fig.1 pComb3 Vector

The basic protocol described here covers the construction of Fab libraries (naïve libraries) in the phagemid vector, pComb3 (Fig.1). This vector has one important feature is that it contains both origin replication of muticopy E.coli plasmid colE1 and the origin of replication of the filamentous bactriohage f1. Furthermore, pComb3 harbors the gene for enzyme β-lactamase that confers ampicillin resistance to bacteria that contain pComb3 DNA. When you design fab library by phage display, you have two choices: 1. Construct library of HC + κ chain or HC + λ chain; 2. Construct library of HC + κ chain + λ chain after mix κ and λ chain. Select appropriate combination of chains according to your need. The basic protocol flow chart is shown in Fig.2.

Basic Protocol of Fab Library Construction Flow Chart Fig.2 Basic Protocol of Fab Library Construction Flow Chart.

Materials

  1. Fresh lymphoid tissue or preparation of lymphocytes for library construction.
  2. Kit for the preparation of mRNA (e.g., an oligo (dT)-purification system).
  3. Kit for the synthesis of cDNA (e.g., “First-strand cDNA synthesis kit”).
  4. PCR reagents: Taq DNA polymerase with 10x Taq DNA polymerase buffer, a stock of deoxyribonucleoside triphosphates (dNTPs). PCR H2O [ACS] reagent;
  5. LC or HC oligonucleotide primers (design based on your requirement).
  6. Kit for the isolation of DNA from gels.
  7. Appropriate restriction enzymes for cloning ( e.g., SacI (100 U/μL), XbaI (100 U/μL), SpeI (50 U/μL), XhoI(40 U/μL), and associated 10X buffers).
  8. Appropriate phagemid vector (e.g., pComb3) (Fig. 1).
  9. Commercial kit for the isolation of plasmid DNA.
  10. Ligation kit, 5x ligation buffer (500 mM Tris-HCl, 25 mM MgCl2, pH7.4).
  11. Electrocompetent Escherichia coli strain (e.g., XL-1 Blue).
  12. Luria-Bertani medium (LB). Super Broth (SB).
  13. Gene Pulser and suitable electroporation cuvets.
  14. 2TY as liquid and solid media.
  15. Glucose 20% (w/v) sterile-filtered.
  16. Filtersterilized antibiotic for screening positive clones (e.g., Ampicillin (100 mg/mL stock) and kanamycin (50 mg/mL stock), tetracycline 50 (μg/mL stock)).
  17. Kit for the purification of PCR products.
  18. QIAquick Gel Extraction Kit.
  19. 20% (w/v) Polyethylene glycol (PEG) 8000, 2.5 M NaCl. Glycerol.
  20. TE buffer (10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid, pH 8.0).

Methods

1. RNA Isolation and cDNA Synthesis

  1. 1.1RNA is extracted from the tissue of interest (e.g., mouse spleen, human lymph node) by using a mRNA preparation kit, and RNA quality is assessed by agarose gel electrophoresis and spectrophotometry. If DNA is present in the RNA sample, then the sample is digested with DNase I. Reverse transcription (RT) of total RNA is done using random primer or Oligo dT by kit for the synthesis of cDNA.
    Note: The RNA method described takes 2 d and gives high-quality RNA. It is suitable for the purification of RNA from small quantities of tissue (>0.05 g) in 2 mL microcentrifuge tubes. For the construction of human libraries, some researchers have found lymph nodes to be a source of RNA superior to peripheral blood lymphocytes. Aseptic technique should be used throughout the procedure and gloves should be worn and changed frequently to minimize RNase contamination. It is best to use disposable plasticware and fresh, sterile solutions. For details on how to treat glassware and nonsterile solutions to prevent contamination with RNases, consult a general laboratory manual. Grinding of sample tissue should be done in a class II Biohazard tissue culture hood. Phenol/chloroform extractions should be performed in a fume hood.

2. Amplification of Light-chain (LC) and Heavy-chain (HC) Genes

  1. 2.1Prepare reaction mix sufficient for all combinations of 3’ and 5’ primers (murine, human, or other species as appropriate, appropriate primers for V-gene families (κ, λ, LCs, and γ HCs)), with PCR reagents. Perform PCR reactions for each gene primers using the optimizing cycling parameters.
  2. 2.2Run 5 μL of each PCR reaction on standard 1% agarose gels to check the size and yield of the products.
  3. 2.3Purify LC and HC PCR products by purification Kit.
    Note: Precautions should be taken when doing PCR to minimize contamination from external sources of template DNA. These are listed as follows: keep cDNA reagents separate from PCR product; PCR reactions are to be set up in a hood or designated bench space; use only PCR-dedicated pipets for setup; wipe down pipets with 0.1 M NaOH, followed by 70% ethanol before use; always use plugged tips and sterile technique so as not to create aerosols that could contaminate other reactions; use sterile disposable plasticware for preparation of reagents and solutions and for PCR reactions; keep caps tightly closed on all tubes not in immediate use; the cDNA should be the last component added to the PCR reaction; pipet PCR product separately, away from reaction assembly area; store PCR products in a dedicated box at -20 °C; negative controls in PCRs are imperative and all primer combinations should be covered.

3. Digestion of LC PCR Product with SacI and XbaI for Cloning into pComb3

  1. 3.1 The LC PCR products are digested with SacI restriction enzyme in a optimized reaction system (e.g., 50 μL system: 1–3μg LC PCR products, SacI (50 U/μg DNA), 10 mM Tris base: Tris-HCl (1_3.5, pH 7.3), 0.1 mg/mL BSA, 7 mM β-mercaptoethanol, 20 mM NaCl, 7 mM MgCl2, the rest is filled with H2O).
  2. 3.2 Purification of the SacI digestion DNA prior to digestion with XbaI restriction enzyme is not necessary. Digest SacI cut DNA product with XbalI in an optimized reaction system.
  3. 3.3 Clean up SacI/XbaI digested LC DNA with commercial DNA purification kit.
    Note: The efficiency of ligation of the LC or HC genes into pComb3 is substantially reduced when incomplete digestion has occurred. To increase the efficiency of digestion of the PCR product with both restriction enzymes, a two-step buffer system was developed. In order to achieve the optimal conditions for digestion of DNA with XbaI, the pH and the salt concentration have to be increased. To do this, the volume of the reaction is doubled as it is for the digestion with XhoI/SpeI of the HC.

4. Digestion of HC PCR Product with SpeI and XhoI

As in Subheading 3.
Note: In some murine HCs, there is an internal XhoI site, which results in additional, smaller products. These need to be gel-purified away from the full-length HC amplicon prior to ligation so that only complete HC genes are cloned into the library.

5. Preparation of Double-digest pComb3 for Cloning of Digested LC Products, and Ligation of LC PCR Products and pComb3

  1. 5.1 pComb3 vector (20–40 μg) (Fig. 1) is digested in a commercial buffer with SacI (50 U/μL) and XbaI (50 U/μL) for 2–3 h at 37 °C. Double-digestion of vector DNA is efficient because of the length of intervening sequence between the restriction sites (compare digestion of PCR products).
  2. 5.2 Run digested products by agarose gel in TBE.
  3. 5.3Purify all digested products from agarose gel with the QIAquick Gel Extraction Kit and determine the DNA concentration through spectrophotometer measurement.
  4. 5.4Ligate gel-purified fragments (LC PCR product) into SacI/XbaI-digested and gel-purified phagemid vector pComb3 by Ligation kit.

6. Electroporation into E. coli XL1 Blue

  1. 6.1 Electroporate the desalted ligation mixtures into competent XL1 Blue according to manufacturer’s recommendations.
  2. 6.2 Spread transformed bacteria on large LB agar plates (24 cm x 24 cm) supplied with 100 mg/ml ampicillin and 1% glucose, and incubate at 37 °C overnight.
  3. 6.3 Isolate plasmid DNA (combination of LC and pComb3) by Commercial kit and determine the DNA concentration.
  4. 6.4Identify insert efficiency by double digestion of SacI/XbaI restriction enzyme.
    Note: A high transformation efficiency of XL1-Blue is required for the construction of Fab libraries. In order to obtain highly electrocompetent cells, work as quickly as possible during resuspension of cells, and do not leave cells on ice any longer than necessary. Cells made competent by chemical means are not of sufficiently high quality for library construction. If electrocompetent cells are not available in-house, a commercial source can be used, although the cost of these can be high.

7. Cloning of HC and Construction of Library Phage

  1. 7.1 Digest plasmid DNA obtained in last procedure with SpeI and XhoI.
  2. 7.2 Ligate digested plasmid DNA product with digested HC product obtained in Subheading 4.
  3. 7.3 Electroporate identified ligation product into competent XL1 Blue (see subheading 6).
  4. 7.4 Add carbenicillin and ampicillin to 20 μg/mL and 100 mg/mL respectively, and incubate a further 60 min at 37 °C.
  5. 7.5 Add 2.5 x 1012 VCSM13 helper phage to each 100 mL culture. Increase the concentration of carbenicillin and ampicillin. Incubate with shaking for 2 h, 37 °C.
  6. 7.6 Centrifuge to collect bacteria and collect the supernatant. Precipitate phage from the supernatant by adding 1/5 vol 2.5 M NaCl, 20% PEG, and incubating on ice for 60 min.
  7. 7.7 Use 50 ml of PBS per liter of culture to resuspend the phage pellet after centrifugation.
  8. 7.8 Repeat the phage precipitation with PEG/NaCl to further purify the phage and resuspend in the same volume of PBS, add glycerol to a final concentration of 50% and freeze at -80 °C 1-ml aliquots for long-term storage as phage library stock.
    NoteInitial stocks of helper phage may need to be purchased, but, afterwards, stocks made in-house are generally of high quality and can be stored for several years at -70 °C without loss of infectivity. Helper phage is stable for a few months at 4 °C. Two-time PEG/NaCl precipitations are needed to further purify the phage library from bacterial contaminates and also soluble antibody fragment or bacterial proteins and possible proteinase contamination.

Here we only provide a basic procedure of fab library construction by phagemid for your reference, although there are numerous examples of different design strategies that have generated functional antibody libraries. Creative Biolabs offers the best service to help researchers make important breakthrough on their complex antibody engineering projects.

If you need help or have any questions about the protocol described above, please feel free to email us at info@creative-biolabs.com or call us on 1-631-871-5806 anytime.

Reference:
  1. P. M. O’Brien and R. Aitken,(2002). Antibody Phage Display.Methods in Molecular Biology.

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